1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleoside linkages of oligonucleotides.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Internucleoside linkages are formed between the 3xe2x80x2 functional group of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide. In the phosphoramidite approach, the internucleoside linkage is a phosphite linkage, whereas in the H-phosphonate approach, it is an H-phosphonate internucleoside linkage. To create the sulfur-containing phosphorothioate internucleoside linkage, the phosphite or H-phosphonate linkage must be oxidized by an appropriate sulfur transfer reagent. In the H-phosphonate approach, this sulfurization is carried out on all of the H-phosphonate linkages in a single step following the completion of oligonucleotide chain assembly, typically using elemental sulfur in a mixed solvent such as CS2/pyridine. In contrast, the phosphoramidite approach allows stepwise sulfurization to take place after each coupling, thereby providing the capability to control the state of each linkage in a site-specific manner.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10xcexcmol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994). Several modifications of the standard phosphoramidite processes have is already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12: 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14: 1349 (1995)) and isolation (Kuijpers et al. Nucl. Acids Res. 18: 5197 (1990); and Reddy et al., Tetrahedron Lett. 35: 4311 (1994)) of oligonucleotides.
In the case of sulfurization, the need for refinement is manifest. Elemental sulfur is not an efficient sulfur transfer reagent due to its poor solubility and slow sulfurization reaction. Consequently, numerous efforts have been made to discover improved sulfur transfer reagents. Kamer et al., Tetrahedron Lett. 30: 6757-6760 (1989) teaches the use of phenylacetyl disulfide as a sulfur transfer reagent. Iyer et al., J. Org. Chem. 55: 4693-4699 (1990) discloses DNA sulfurization using 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent). Vu et al., Tetrahedron Lett. 32: 3005-3008 (1991) teaches sulfur transfer using tetraethylthiuram disulfide (TETD). Rao et al., Tetrahedron Lett. 33: 4839-4842 (1992) discloses dibenzoyl tetrasulfide as a sulfurizing agent. Stec et al., Tetrahedron Lett. 33: 5317-5320 (1993) teaches DNA sulfurization using bis(O,O,-diisopropoxyphosphinothioyl) disulfide (S-Tetra). Rao et al., Tetrahedron Lett. 35: 6741-6744 (1994) discloses benzyltriethyl-ammonium tetrathiomolybate (BTTM) as a sulfur transfer reagent. Effimov et al., Nucleic Acids Res. 23: 4029-4033 (1995) teaches the use of bis(p-toluenesulfonyl) disulfide for sulfur transfer. Xu et al., discloses 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) and 1,2,4-dithiazolidine-3,5-dione (DTSNH) as sulfur transfer reagents. Unfortunately, each of these reagents has its limitations and only Beaucage reagent and TETD have become commercially available, with only Beaucage reagent being widely used. Moreover, Beaucage reagent has sub-optimal synthesis and stability characteristics and during sulfurization forms a by-product, 3H-2,1-benzoxathiolan-3-one-1-oxide, which is a powerful oxidizing agent capable of producing unwanted phosphodiester internucleoside linkages.
There is, therefore, a need for new sulfur transfer reagents and processes for sulfurizing oligonucleotides. Ideally, such reagents should be inexpensive to make, stable in storage and highly efficient in sulfurization.
The invention provides new sulfur transfer reagents and processes for their use in sulfurizing oligonucleotides. The sulfur transfer reagents according to the invention are inexpensive to make, stable in storage and highly efficient in sulfurization.
In a first aspect, the invention provides novel sulfur transfer reagents having the general structure: 
wherein each R is independently a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxy, or amino groups. In a particularly preferred embodiment, each R is an ethyl group.
In a second aspect, the invention provides novel processes for adding a sulfur group to an internucleoside linkage of an oligonucleotide using the novel sulfur transfer reagents according to the invention. In preferred embodiments, the novel processes according to the invention comprise contacting an oligonucleotide having at least one sulfurizable internucleoside linkage with a novel sulfur transfer reagent according to the invention for a time sufficient for sulfurization of the sulfurizable internucleoside linkage(s) to occur.